Trizol

qRT-PCR was used to detect the RNA expression of METTL14 and miR-1306-5p. All the RNA was collected from blood, cell, or tissue samples using the miRNeasy extraction kit (QIAGEN). For quantitative analysis, the cDNA was reversed by miRNA Reverse Transcription Kit (MR101-01/02, Vazyme) and detected by all-in-one miRNA RT-qPCR Detection Kit (Q711-02, Vazyme) with U6 as the internal control. As for METTL14 mRNA detection, TRIzol (BS259A, Biosharp) was used for RNA isolation and PrimeScript RT Reagent kit (R223-01, Vazyme) was used to reverse RNA into cDNA. SYBR Green Real-Time PCR Master Mix (Q711-02, Vazyme) was used for RT-PCR assay with β-actin as the control. The primers for miR-1306-5p, U6, METTL14, and β-actin were listed as below: METTL14, sense, 5′- GAGTGTGTTTACGAAAATGGGGT-3′; antisense, 5′- CCGTCTGTGCTACGCTTCA-3′; β-actin: sense, 5′-AGCGAGCATCCCCCAAAGTT-3′, antisense: 5′-GGGCACGAAGGCTCATCATT-3′; U6: sense, 5′-CTCGCTTCGGCAGCACA-3′, antisense: 5′-AACGCTTCACGAATTTGCGT-3′; miR-1306-5p reverse primer: 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTGGACGTT-3′; miR-1306-5p sense: 5′-AATACCACCTCCCCTGCA-3′. 2^−ΔΔCt method was used for analysis of relative expression.

Total RNA was isolated from sample heart tissues with Trizol (Biosharp) according to the manufacturer’s instructions. cDNA was synthesized with HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme). The ChamQ Universal SYBR qPCR Master Mix (Vazyme) was used in quantitative real-time PCR (qRT-PCR) with Quant Gene 9600 system (Bioer Technology). β-actin was used as internal control to calculate the relative mRNA expression in each sample by the 2^−∆∆Ct method. The primer sequences (Generay Biotech) of each gene were listed in Table 1.
Table 1

The primer sequences used for analysis

Genes	Primer sequences
Bgn	R: TGTGCTACTCACCTTGCTG
	F: TGGTGTCTGAACCTGAAGCC
Ctgf	R: TGCTGTGCATCCTCCTACCG
	F: CAGAGAGCGAGGAGCACCAA
Col5a2	R: ATGGGGTATGTAAAGCCTCAGC
	F: AGGACACAGGGGGCATTACT
Eln	R: CTGACTCGCGACCTAATCAA
	F: TCCTTGTCCTGTGGGTTTCC
Sox9	R: GGAAGGTAACGATTGCTGGG
	F: CCCTCCTCGCTGATACTGGT
Tgfb3	R: TGATGACCCACGTCCCCTAT
	F: CAGACTCCGAGGTCTCCTGA
Postn	R: GCAAACCACTTTCACCGACC
	F: CGTTGGTCCATGCTCAGAGT
Tgfb2	R: GCCAGGACACGAAAATCACG
	F: TCCCTCCCTCCTGTCACAAA
β-actin	R: GGGCAACCTTCCCAATAAAT
	F: CCATGTTCCAAAACCATTCC

The total RNA of MLO-Y4 cells was extracted by TRIzol (Biosharp), and cDNA was reverse-transcribed using HiScript 1st Strand cDNA Synthesis Kit (Vazyme) and real-time PCR using SYBR qPCR Mix (Vazyme). The primer sequences were as follows: β-actin (mouse): 5′-CATTGCTGACAGGATGCAGAAGG-3′ (forward) and 5′-TGCTGGAAGGTGGACAGTGAGG-3′ (reverse); IL-6 (mouse): 5′-TACCACTTCACAAGTCGGAGGC-3′ (forward) and 5′-CTGCAAGTGCATCATCGTTGTTC-3′ (reverse); P53 (mouse): 5′-CCTCAGCATCTTATCCGAGTGG-3′ (forward) and 5′-TGGATGGTGGTACAGTCAGAGC-3′ (reverse); P21 (mouse): 5′-TCGCTGTCTTGCACTCTGGTGT-3′ (forward) and 5′-CCAATCTGCGCTTGGAGTGATAG-3′ (reverse); P27 (mouse): 5′-AGCAGTGTCCAGGGATGAGGAA-3′ (forward) and 5′-TTCTTGGGCGTCTGCTCCACAG-3′ (reverse); and Opg (mouse): 5′-CGGAAACAGAGAAGCCACGCAA-3′ (forward) and 5′-CTGTCCACCAAAACACTCAGCC-3′ (reverse).